The interaction of sugar and protein (hereinafter “sugar-protein interaction”) is an area of study that has been looked at with interest in structural biology for better understanding of cell-cell, cell-extracellular matrix, and cell-pathogen interactions.
Generally, sugar-protein interaction is analyzed through labeling of ligand proteins or conjugate sugars with radioactive isotopes or enzymes. For example, in ELISA or blotting methods, there have been techniques that use sugar chains labeled by pyridyl amination or fluorescence, or proteins labeled with secondary antibodies. As a method that analyzes sugar-protein interaction using sugar chains labeled by pyridyl amination or fluorescence, there is a method in which fluorescent sugar chains with pyridylamine are used to measure sugar-protein interaction by TLC (thin layer chromatography) or HPLC (high-pressure liquid chromatography) (Non-Patent Publication 1). As a method that analyzes sugar-protein interaction using proteins that have been labeled with secondary antibodies or the like, there is a method that uses labeled secondary antibodies to measure fluorescence in arrays (Non-Patent Publication 2).
As examples of a method that does not use a label, there have been available surface plasmon resonance (hereinafter, “SPR”) and quarts crystal microbalance (hereinafter, “QCM”).
SPR is a method in which a ligand complex including an oligosaccharide that mimics part of the sugar is introduced onto a surface of a sensor chip, which is then used to specify substances, such as proteins, that specifically interact with the oligosaccharide. QCM measures sugar-protein interaction by measuring changes in oscillation frequency of the crystal oscillator disturbed by adhesion of some material, by taking advantage of the characteristics of the crystal oscillator that the oscillation of the crystal oscillator under AC voltage remains constant unless it is disturbed by external factors.
There have been many reports concerning synthesis techniques for the immobilization of sugars, including a method in which a sugar chain modified with an aromatic amino group is bound to an aldehyde group in a metal colloid by reductive amination (Non-Patent Publication 3), and a method in which a compound with a thiol group for conjugation with a metal colloid is bound to a sugar chain by glycosylation (trichloroacetimidate method) (Non-Patent Publication 4)
[Non-Patent Publication 1]
M. S. Stoll et al., Eur. J. Biochem. 267, 1795-1804, 2000
[Non-Patent Publication 2]
M. Schwarz et al., Glycobiol. 13, no. 11, 749-754, 2003
[Non-Patent Publication 3]
H. Otsuka et al., J. Am. Chem. Soc. 123, 8226-8230, 2001
[Non-Patent Publication 4]
J. M. de la Fuente et al., Angew. Chem. Int. Ed. 40, No. 12, 2258-2260, 2001
A problem of the method that measures sugar-protein interaction using labeled sugar chains or proteins, however, is that it requires labeling as a pre-treatment and the effect of labeling greatly influences measurement variations. Further, while non-labeling methods have been available, the SPR or QCM requires very expensive equipment, which has made it difficult to perform measurement conveniently.
Further, it has been difficult to readily immobilize sugar chains due to the very complicated procedures required for the synthesis and purification, as exemplified by the method of Non-Patent Publication 3 which requires a pre-treatment for the sugar chain that has incorporated therein an aromatic amino group, and by the method of Non-Patent Publication 4 which requires at least five steps for the synthesis.
Further, while the method of evaluating sugar chains based on protein has been used for some time as a technique of analyzing sugar-protein interaction, it is not common to evaluate proteins based on sugar chain. Further, while it would be highly economical, no method has been available that enables a protein to be recovered from a sugar-protein interactant formed by the interaction of sugar and protein.
The present invention was made in view of the foregoing problems, and an object of the invention is to provide a novel sugar-immobilized metal particle that is stable and can readily immobilize a sugar chain, a method for measuring sugar-protein interaction both easily and inexpensively without labeling, using the sugar-immobilized metal particle, and a method for conveniently recovering a protein from a sugar-protein interactant.